US11067579B2 - Target marker GP73 for detecting steatohepatitis and detection application method - Google Patents
Target marker GP73 for detecting steatohepatitis and detection application method Download PDFInfo
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- US11067579B2 US11067579B2 US16/638,713 US201716638713A US11067579B2 US 11067579 B2 US11067579 B2 US 11067579B2 US 201716638713 A US201716638713 A US 201716638713A US 11067579 B2 US11067579 B2 US 11067579B2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
Definitions
- the present invention relates to a novel marker and a detection method for detecting steatohepatitis in vitro, and belongs to the subfield of biotechnology and in vitro diagnosis.
- Fatty liver refers to a disease in which there is excessive abnormal accumulation of fat in liver cells due to various reasons.
- Normal human liver tissue contains a small amount of fat, and the weight thereof accounts for about 4% to 5% of the weight of the liver. If the amount of fat exceeds 5%, it is mild fatty liver; if the amount of fat exceeds 10%, it is moderate fatty liver, and if the amount of fat exceeds 25%, it is severe fatty liver.
- Fatty liver is currently one of the most common chronic liver diseases, with an incidence rate of more than 30% in developed countries and some developing countries. It has become an important public health problem and seriously threatens the health of people.
- Fatty liver includes the main pathological stages of a simple fatty liver, steatohepatitis, liver fibrosis, liver cirrhosis and so on; among those stages of fatty liver, a simple fatty liver is benign, and can be reversed through diet, exercise regulation and the like, without the need for specific treatment; however, if steatohepatitis is not treated in time, it will develop into liver fibrosis, liver cirrhosis, and liver cancer as the disease progresses; it is reported that the incidence rate of steatohepatitis converting to liver fibrosis, liver cirrhosis, finally to liver cancer is equivalent to the incidence rate of viral hepatitis.
- Steatohepatitis is a serious threat to the health of people.
- fatty liver is mainly detected by B-mode ultrasound, CT, MM and other imaging means, but imaging detection cannot distinguish between a simple fatty liver and steatohepatitis.
- the diagnosis of steatohepatitis still depends on liver puncture detection, which will bring great physical and psychological burden to patients, and is not conducive to the diagnosis and daily detection of steatohepatitis.
- Golgi protein 73 (hereinafter referred to as GP73 unless otherwise specified) is a Golgi glycoprotein, and a novel transmembrane glycoprotein first discovered in 2000 by Kladney et al. in the study of the etiology of adult giant cell hepatitis. It is also known as type II Golgi membrane protein (Golph 2) and Golgi membrane protein I (Golm 1), and has a relative molecular weight of 73,000 kDa. GP73 is not expressed or expressed in a small amount in normal liver cells.
- GP73 is highly expressed in a variety of liver diseases such as liver fibrosis, liver cirrhosis, and liver cancer, and can be secreted into serum; an increase of GP73 is most obvious in patients with liver cancer, so GP73 is one of the preferred markers for early diagnosis of liver cancer, and the sensitivity and specificity thereof are better than those of AFP.
- Chinese Patent Publication CN 101735319 A (BEIJING HOTGEN BIOTECH CO LTD, patent name “Monoclonal antibody against GP73 protein, preparation method and application thereof”) discloses a monoclonal antibody against Golgi protein 73, i.e., GP73, and an enzyme-linked immunological quantitative detection kit and a rapid diagnostic kit employing advantage of GP73; and discloses that the critical value of GP73 content for distinguishing between normal people and patients with liver diseases is 40 ng/ml, and that the critical value of GP73 content for distinguishing between patients with benign liver diseases and patients with malignant liver diseases is 100 ng/ml.
- Patent Name “Anti-Golgi apparatus protein monoclonal antibody and use” discloses an anti-GP73 monoclonal antibody, an enzyme immunoreagent for liver cancer detection using the monoclonal antibody, and applications thereof in immunoprecipitation, immunoblotting, immunohistochemistry and the like.
- Chinese Patent Publication CN 104215761 A discloses an enzyme-linked immunoassay kit for detecting an anti-GP73 antibody in serum, which is mainly used for liver cancer detection.
- the present invention provides a novel serological target marker GP73 for diagnosing and identifying a simple fatty liver and steatohepatitis in populations with fatty liver, and further provides a detection kit for detecting GP73, a detection method and the determination criteria thereof for diagnosis and identification of a simple fatty liver and steatohepatitis in populations with fatty liver.
- the present invention provides a novel serological target marker GP73 for diagnosing and identifying a simple fatty liver and steatohepatitis in populations with fatty liver, and an in vitro diagnosis method for detecting the content of GP73 in test samples.
- the present invention further provides a serological in vitro diagnosis detection kit for diagnosing and identifying a simple fatty liver and steatohepatitis in populations with fatty liver, a method for detecting GP73 by using the in vitro diagnosis detection kit, and the determination criteria thereof.
- the principle of the GP73 detection method is the principle of immunological detection methods based on an antigen-antibody binding reaction, and the method is preferably any one of chemiluminescence, enzyme-linked immunoassay, and immunochromatography.
- the chemiluminescence is at least one of chemiluminescence, magnetic particle-based chemiluminescence, electrochemiluminescence, enzymatic chemiluminescence, and time-resolved chemiluminescence.
- the enzyme-linked immunoassay is at least one of enzyme-linked immunosorbent assay, immunofiltration assay, and protein chip.
- the immunochrom atography is at least one of up-conversion luminescence immunochromatography, latex immunochromatography, colloidal gold immunochromatography, luminescence immunochromatography, and quantum dot immunochromatography.
- the GP73 test sample is a blood sample from populations with fatty liver, and the test result of the blood sample is used for diagnosing and identifying a simple fatty liver and steatohepatitis in populations with fatty liver.
- the determination cutoff value of the GP73 serological in vitro diagnosis detection kit for diagnosing and identifying a simple fatty liver and steatohepatitis in populations with fatty liver is 82.25 ng/ml.
- the present invention provides a novel serological target marker GP73 for diagnosing and identifying a simple fatty liver and steatohepatitis in populations with fatty liver, and further provides a GP73 detection method for diagnosing and identifying a simple fatty liver and steatohepatitis in populations with fatty liver, and the determination criteria thereof, which solve the problem that the existing imaging and serological detection techniques cannot diagnose and identify a simple fatty liver and steatohepatitis in populations with fatty liver, and provide a basis for diagnosis and treatment of steatohepatitis; the present invention adopts a blood sample as the test sample, which greatly reduces the pain of patients caused by detecting steatohepatitis through liver puncture; and thus the present invention has important clinical diagnostic value and clinical significance for identifying a simple fatty liver and steatohepatitis in populations with fatty liver.
- FIG. 1 is a diagram showing the absolute bias between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent.
- FIG. 2 is a diagram showing the absolute bias between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- FIG. 3 is a diagram showing the absolute bias between the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- FIG. 4 is a diagram showing the relative bias between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent.
- FIG. 5 is a diagram showing the relative bias between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- FIG. 6 is a diagram showing the relative bias between the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- FIG. 7 shows a linear correlation between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent.
- FIG. 8 shows a linear correlation between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- FIG. 9 shows a linear correlation between the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- FIG. 10 is a ROC curve analysis chart.
- FIG. 11 is a diagram showing the data distribution of GP73 detection results.
- FIG. 12 is a clinical consistency analysis chart.
- the present invention provides a GP73 target marker, a method for detecting GP73 and the determination criteria thereof, wherein the anti-GP73 protein antibody was applied to prepare a detection reagent working on the principle of immunological detection methods based on an antigen-antibody binding reaction, including a quantitative detection reagent of any one of chemiluminescence, enzyme-linked immunoassay, immunochromatography and methods improved based on the foregoing methods;
- the sample detected by using the quantitative reagent is a blood sample from populations with fatty liver, and may be any one of serum, plasma, and whole blood;
- the detection result of the blood sample is used for diagnosing and identifying a simple fatty liver and steatohepatitis in populations with fatty liver.
- the method of measuring GP73 in the present invention includes using reagents including, but not limited to, antibodies.
- the antibodies specifically include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments, such as variable domains and other portions in antibodies that exhibit desired biological activity.
- the term “monoclonal antibody (mAb)” refers to an antibody that is highly specific and is directed against a single antigenic determinant (epitope). Thus, the term “monoclonal” refers to antibodies directed against the same epitope, and should not be construed as the production of the antibodies requiring any particular method.
- monoclonal antibodies can be prepared by any technique or method known in the art, including, for example, the hybridoma method (Kohler et al., 1975, Nature 256: 495), or the recombinant DNA method known in the art (for example, see U.S. Pat. No. 4,816,567), or a method for producing monoclonal antibodies in a recombinant manner by using a phage antibody library and by using the techniques described in the following literature for separation: Clackson et al., 1991, Nature 352: 624-628; and Marks et al., 1991, J. Mol. Biol. 222: 581-597.
- the “antibody fragment” refers to a molecule different from an intact antibody, which comprises a portion of the intact antibody and binds to an antigen to which the intact antibody binds.
- antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, and F(ab′)2; diabodies; linear antibodies; single chain antibody molecules (e.g., scFv); and multi-specific antibodies formed from antibody fragments.
- the reagents according to the present invention also include other reagents.
- the other reagents include an ABC diluent, a TMB chromogen solution, a TMB stop solution, a wash solution, and the like.
- any one of the above substances may be stored separately in different containers (e.g., vials) in a state of being separated from the other substances, as long as they can be in contact with each other at the time of use.
- any two or more of the above substances may be mixed to exist as a mixture.
- the other reagents may be provided respectively in the form of a dry powder, or in the form of a solution, such as in the form of an aqueous solution.
- concentration or content of each of these substances can be easily determined by those skilled in the art according to different needs.
- the concentration of the substance may be high.
- the concentration may be reduced to a working concentration by, for example, diluting the above-mentioned solution with a high concentration.
- a reagent for measuring GP73 may be further prepared as a diagnostic agent for identifying a simple fatty liver and steatohepatitis in a subject with fatty liver.
- the diagnostic agent may be in the form of a diagnostic composition, a diagnostic kit, or any other forms in which a plurality of reagents that are present separately are used in combination.
- anti-GP73 protein antibody was prepared with reference to existing patented technologies, or selected from commercially available ones, or prepared by generally known technologies, and the prepared or selected monoclonal antibody can be used for accurate quantitative detection and determination of GP73 in blood samples from populations with fatty liver.
- the anti-GP73 protein antibody described in certain embodiments of the present invention is preferably the anti-GP73 protein antibody prepared by the anti-GP73 protein antibody preparation method in the patented technology of BEIJING HOTGEN BIOTECH CO LTD, CN 101735319 B, “Monoclonal antibody against GP73 protein, preparation method and application thereof”, and the hybridoma cell line (3E12 strain) for preparing the anti-GP73 protein antibody was deposited under the deposit number CGMCC No. 2742 on Nov. 11, 2008 in the China General Microbiological Culture Collection Center (CGMCC).
- CGMCC China General Microbiological Culture Collection Center
- mice were inoculated intraperitoneally with pristane or liquid paraffin, 0.3 to 0.5 ml per mouse; 7 to 10 days later, the mice were inoculated intraperitoneally with the hybridoma cell line, 3E12 strain which had been diluted with PBS or serum-free medium, 5 ⁇ 10 5 /0.2 ml per mouse; ascitic fluid production in the mice was observed every day; if the abdomens were significantly enlarged and the skins felt tense when touched by hand, ascitic fluid could be collected, about 3 ml of ascitic fluid was collected per mouse; the ascitic fluid was centrifuged at 2000 r/min for 5 minutes to remove cell components and other precipitates; the supernatant was collected and purified by a recombinant protein G pre-packed chromatography column to obtain the anti-GP73 monoclonal antibody.
- the chemiluminescence is at least one of chemiluminescence, magnetic particle-based chemiluminescence, electrochemiluminescence, enzymatic chemiluminescence, time-resolved chemiluminescence, and improved methods thereof.
- Those different chemiluminescence methods are different only in labeling particle and separation method, but they share the same reaction principle.
- anti-GP73 protein antibody concentration optimization treatment those methods all can realize accurate quantitative detection and identification of a simple fatty liver and steatohepatitis in populations with fatty liver, and achieve the purpose of auxiliary diagnosis and treatment of steatohepatitis.
- the preparation of those GP73 chemiluminescence methods comes from mature chemiluminescence preparation technologies, and those technologies were optimized to achieve excellent detection results.
- the chemiluminescence used for preparing the chemiluminescence detection reagent was preferably magnetic particle-based chemiluminescence
- the magnetic particle-based chemiluminescence detection reagent was prepared with reference to the method disclosed in the related patented technology of BEIJING HOTGEN BIOTECH CO LTD
- the antibody was the anti-GP73 protein antibody described in Example 1.
- the preparation of the GP73 separation reagent was as follows: magnetic particles were reacted with the anti-GP73 protein antibody to form a conjugate, which was added at a certain concentration into a buffer to prepare the separation reagent.
- the main steps were as follows:
- the coating buffer was a 0.1 mol/L MES buffer at pH 5.0.
- the final concentration of the activator carbodiimide was 0.01 to 0.1 g/ml.
- the appropriate concentration of the anti-GP73 protein antibody was 10 to 100 ug/mg magnetic beads.
- the main components of the blocking buffer included a 50 mM Tris buffer, a 1 to 5% BSA solution, and a 0.1% preservative, and the pH of the blocking buffer was 7.4.
- the main components of the wash solution included a 0.02 M PBS, a 100 nM sodium chloride solution, and a 0.5 to 1% TWEEN®-20 solution (Polysorbate 20).
- a magnetic field was added to discard the supernatant, and a magnetic particle preservation liquid having a volume of exact 10 times the volume of the mixed solution was added to preserve the magnetic particles of the anti-DCP monoclonal antibody, thereby giving the GP73 separation reagent.
- the main components of the preservation liquid included a 0.02 to 0.1 M PBS solution, a 1% to 5% BSA solution, a 0.1% preservative, and a 0.5 to 3% glycine solution, and the pH of the preservation liquid was 7.4.
- the preparation of the detection reagent was as follows: an alkaline phosphatase-labeled anti-GP73 protein antibody was added at a certain concentration into a buffer to prepare the detection reagent.
- the alkaline phosphatase-labeled antibody was preferably an anti-GP73 protein antibody; and the preparation method was merely illustrative of the present invention, and should not be considered as a limitation on the preparation and use of the labeled antibody.
- Alkaline phosphatase was taken so that the mass ratio of alkaline phosphatase to anti-GP73 protein antibody was 1/4 to 1/1, NaIO 4 (12.8 mg/ml, 4° C.) having the same volume as that of the alkaline phosphatase solution was added, and the reaction took place in the dark at 4° C. for 30 minutes; then an ethylene glycol solution having the same volume as that of the alkaline phosphatase solution was added, and the reaction took place in the dark at 4° C.
- the alkaline phosphatase-labeled anti-GP73 protein antibody was added in a certain ratio (recommended concentration ⁇ 1:1000) into the preservation liquid, and the solution was mixed uniformly to give the GP73-labeled antibody, which is the prepared detection reagent.
- the main components of the wash solution included a 0.1 M Tris buffer, a 0.1 to 1% TWEEN® solution (Polysorbate 20), and a 0.1% preservative.
- the preparation of the GP73 calibration solution was as follows: the GP73 antigen that passed inspection was added into a calibration production diluent, and the resultant was lyophilized to prepare the calibration solution.
- the calibration production diluent included a 1 to 5% BSA solution, a 1 to 5% trehalose solution, and a 10 to 50% newborn bovine serum solution.
- the high-value GP73 antigen lyophilized solution was subjected to gradient dilution until it was diluted to an appropriate concentration. After detection, it was placed in a lyophilizer for lyophilization, wherein the pre-freezing time was 2 hours and the vacuumizing time was 16 to 24 hours.
- the GP73 separation reagent, the GP73 detection reagent, the GP73 wash solution, and the GP73 substrate solution were respectively packaged into reagent bottles of corresponding size, or into the corresponding hole positions in the reagent strip of a strip-packaged reagent, and then sealed.
- the respectively packaged reagents, together with the calibration solution, a consumable reaction tank, a disposable sampling head, etc. constitute the GP73 magnetic particle-based chemiluminescence detection reagent, and the reagent is suitable for use with the automatic immunoassay analyzer of MQ60 series of BEIJING HOTGEN BIOTECH CO LTD.
- the enzyme-linked immunoassay is at least one of enzyme-linked immunosorbent assay, dot immunofiltration assay, magnetic particle-based enzyme-linked immunoassay, enzyme-linked immunofluorescence measurement, and protein chip, or at least one of the improved methods of the corresponding enzyme-linked immunoassays.
- Those different enzyme-linked immunoassays are different only in carrier, but they share the same reaction principle.
- those methods After being subjected to anti-GP73 protein antibody concentration optimization treatment, those methods all can realize accurate quantitative detection and identification of a simple fatty liver and steatohepatitis in populations with fatty liver, and achieve the purpose of auxiliary diagnosis and treatment of steatohepatitis.
- the preparation of those GP 73 enzyme-linked immunoassays comes from mature enzyme-linked immunoassay preparation technologies, and those technologies were optimized to achieve excellent detection results.
- the enzyme-linked immunoassay used for preparing the reagent was preferably enzyme-linked immunosorbent assay
- the detection reagent prepared by enzyme-linked immunoassay was prepared with reference to the method disclosed in the related patented technology of BEIJING HOTGEN BIOTECH CO LTD
- the antibody was the anti-GP73 protein antibody described in Example 1.
- the GP73 enzyme-linked immunoassay detection reagent (for 96 persons) consists of 5 bottles of GP73 standard; 1 anti-GP73 protein antibody coated plate (96 wells); 1 bottle of a horseradish peroxidase (HRP) labeled anti-GP73 protein antibody, 10 ml/bottle; 1 bottle of a chromogen solution A and 1 bottle of a chromogen solution B, 5 ml/bottle for each solution; 1 bottle of a stop solution, 5 ml/bottle; and 1 bottle of a wash solution (20 times concentrated), 50 ml/bottle.
- HRP horseradish peroxidase
- the high-value GP73 antigen was diluted in a 2% BSA solution at different concentrations of 0, 20 ng/ml, 40 ng/ml, 100 ng/ml, and 250 ng/ml; the preservative procline 300 was added to reach a final concentration of 0.1%; the resultant solutions were filtered and sterilized, and respectively placed into 1.5 ml vials, 0.5 ml per vial under sterile conditions. And they were stored at 4° C.
- the ELISA plate was a 12 ⁇ 8 or 8 ⁇ 12 detachable ELISA strip plate.
- the anti-GP73 protein antibody was diluted to 10 to 50 ⁇ g/ml with a 0.05 mol/L, pH 9.5 carbonate buffer, and thereafter the resultant solution was added into each well of the ELISA plate, 100 ⁇ l per well; adsorption was performed overnight; the plate was washed with a wash buffer (a PBST solution containing a 20 mmol/L PBS and a 0.5% TWEEN®-20 (Polysorbate 20), the pH of the wash buffer was 7.4), and then each well was blocked with 120 ⁇ l of blocking buffer (a 2% BSA, diluted in a PBST solution) overnight; the blocking buffer was discarded, and the plate was air-dried to obtain an anti-GP73 protein antibody coated plate (96 wells). The plate was vacuum-sealed and packaged with an aluminum foil bag for later use.
- the anti-GP73 protein antibody concentration was adjusted to about 5 mg/ml; the anti-GP73 protein antibody was mixed with HRP at a ratio of 1:4; and the mixture was dialyzed in a 50 mmol/L, pH 9.5 carbonate buffer for more than 6 hours, wherein the buffer was changed after the first two hours.
- the reaction was terminated with freshly prepared 1 mg of a NaBH 4 solution, the resultant solution was shaken well, and left at 4° C. to stand for 2 hours; thereafter, the solution was shaken every half hour.
- the solution was dialyzed overnight with a 10 mM, pH 7.2 PBS (0.01 mol/L Na 2 HPO 4 and NaH 2 PO 4 stock solutions were prepared in advance, and the two solutions were mixed into a PBS buffer according to the required pH), and the PBS was changed once.
- the anti-GP73 protein antibody solution for which the HRP labeling had been completed was dialyzed overnight with a saturated ammonium sulfate solution, and then centrifuged at 12,000 rpm in an ultrafiltration tube to obtain a concentrated and purified HRP-labeled anti-GP73 protein antibody.
- the HRP-labeled anti-GP73 protein antibody was diluted to a suitable working concentration with a buffer (20 mmol/L PBS) containing 10% fetal bovine serum. The recommended dilution concentration was ⁇ 1:500. The resultant solution was divided and placed into bottles, 10 ml/bottle, and stored at 4° C.
- chromogen solution A a phosphoric acid-citric acid buffer (50 mmol/L, pH 5.0) and a 3% hydrogen peroxide solution were used to prepare the solution, and the solution was divided and placed into bottles, 5 ml/bottle.
- chromogen solution B a TMB (0.1 mg/ml) methanol solution divided and placed into bottles, 5 ml/bottle.
- stop solution 2 mol/L H 2 SO 4 , divided and placed into bottles, 5 ml/bottle.
- wash solution (20 times concentrated solution): a PBS (pH 7.4) and a 1% TWEEN®-20 (Polysorbate 20) solution were used to prepare the solution, and the solution was divided and placed into bottles, 50 ml/bottle.
- a PBS pH 7.4
- a 1% TWEEN®-20 Polysorbate 20
- the GP73 standard, the anti-GP73 protein antibody coated plate, the HRP-labeled anti-GP73 protein antibody, the chromogen solution A, the chromogen solution B, 1 bottle of the stop solution, and the wash solution (20 times concentrated) were assembled as required into the GP73 enzyme-linked immunoassay detection reagent (for 96 persons).
- the method of using the GP73 enzyme-linked immunoassay detection reagent included the following main steps:
- wash buffer The concentrated buffer was diluted 20 times with purified water for later use.
- the standards included 5 different concentrations, which were respectively 0, 20 ng/ml, 40 ng/ml, 100 ng/ml, and 250 ng/ml.
- Antigen-antibody reaction 100 ⁇ l of sample diluent was added into each micro-well of the antibody coated plate, and then 10 ⁇ l of respective standards at different concentrations or the serum samples to be tested were added and incubated at 37° C. for 30 minutes; and the plate was then washed 4 times with the wash buffer.
- Enzyme-linked reaction 100 ⁇ l of the HRP-labeled anti-GP73 protein antibody solution was added to each well and incubated at 37° C. for 30 minutes, and then the plate was washed 4 times with the wash buffer.
- the immunochromatography is at least one of up-conversion luminescence immunochromatography, latex immunochromatography, colloidal gold immunochromatography, luminescence immunochromatography, and quantum dot immunochromatography.
- the immunochromatography is at least one of up-conversion luminescence immunochromatography, latex immunochromatography, colloidal gold immunochromatography, luminescence immunochromatography, quantum dot immunochromatography, and improved methods of the corresponding immunochromatography methods.
- Those different immunochromatography methods are different only in labeling particle, but they share the same reaction principle.
- anti-GP73 protein antibody concentration optimization treatment those methods all can realize accurate quantitative detection and identification of a simple fatty liver and steatohepatitis in populations with fatty liver, and achieve the purpose of auxiliary diagnosis and treatment of steatohepatitis.
- the preparation of those GP 73 immunochromatography methods comes from mature immunochromatography preparation technologies, and those technologies were optimized to achieve excellent detection results.
- the immunochromatography used for preparing the detection reagent was preferably up-conversion luminescence immunochromatography, and the detection reagent prepared by up-conversion luminescence immunochromatography was prepared with reference to the method disclosed in the related patented technology of BEIJING HOTGEN BIOTECH CO LTD, and the antibody was the anti-GP73 protein antibody described in Example 1.
- the anti-GP73 protein antibody was diluted to 2 mg/ml to serve as a T-line coating solution, and the goat anti-mouse IgG was diluted to 2 mg/ml to serve as a C-line coating solution.
- the T-line coating solution and the C line coating solution were coated on a nitrocellulose membrane by a film sprayer, and the membrane was air-dried to obtain a monoclonal antibody coated membrane strip.
- nitrocellulose membrane, an absorbent paper, the bonding pad, and a sample pad were stuck in this order onto a bottom plate, and cut into 4.0 mm wide strips; then the strips were put into the bottom case of a plastic card, covered with the top case of the plastic card, and pressed firmly; desiccant was added and the product was sealed and packaged.
- the components of the sample diluent included a 0.20 M PB solution at pH 7.2, and a 1% TWEEN®-20 (Polysorbate 20) added thereto.
- a total of 200 samples were clinically diagnosed with fatty liver backgrounds, including 63 serum/plasma samples which were liver disease progression samples diagnosed with steatohepatitis backgrounds (including liver fibrosis samples and liver cirrhosis samples caused by steatohepatitis, but not including samples from liver cancer patients; hereinafter simply referred to as steatohepatitis samples) and 137 fatty liver samples with a simple fatty liver.
- Whole blood samples are not easy to be stored for long periods of time, and it is not suitable to establish long-term cohort samples for detection and analysis. No analysis of whole blood samples is made in the Examples described in the present invention, but the methods for detecting GP73 and the determination criteria thereof, which are suitable for GP73 detection in whole blood and improved based on the present invention, used for diagnosis and identification of a simple fatty liver and steatohepatitis in populations with fatty liver, are still considered to be within the scope of the present invention.
- the quantitative detection reagents of three methods including the GP73 magnetic particle-based chemiluminescence detection reagent, the GP73 enzyme-linked immunoassay detection reagent, and the GP73 immunochromatography detection reagent were respectively used to detect 200 samples clinically diagnosed with fatty liver backgrounds, and the detection results were respectively summarized and analyzed.
- the absolute bias, the relative bias, and the linear correlation of the detection results respectively obtained by using the three reagents including the GP73 magnetic particle-based chemiluminescence detection reagent, the GP73 enzyme-linked immunoassay detection reagent, and the GP73 immunochromatography detection reagent were statistically analyzed to determine deviation among those different methodologies.
- the maximum absolute deviation between the detection results of the 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and those obtained by using the GP73 enzyme-linked immunoassay detection reagent was 20.08, which was less than 4 times of the average absolute deviation between the detection results of the 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and those obtained by using the GP73 enzyme-linked immunoassay detection reagent, namely, 20.22.
- FIG. 1 a diagram showing the absolute bias between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent.
- the maximum absolute deviation between the detection results of the 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and those obtained by using the GP73 immunochromatography detection reagent was 20.92, which was less than 4 times of the average absolute deviation between the detection results of the 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and those obtained by using the GP73 immunochromatography detection reagent, namely, 20.10.
- FIG. 2 a diagram showing the absolute bias between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- the maximum absolute deviation between the detection results of the 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent and those obtained by using the GP73 immunochromatography detection reagent was 17.43, which was less than 4 times of the average absolute deviation between the detection results of the 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent and those obtained by using the GP73 immunochromatography detection reagent, namely, 19.03.
- FIG. 3 a diagram showing the absolute bias between the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- the absolute deviations among the detection results of the three reagents including the GP73 magnetic particle-based chemiluminescence detection reagent, the GP73 enzyme-linked immunoassay detection reagent, and the GP73 immunochromatography detection reagent all fell within the range specified by NCCLS EP9-A, so there was no significant deviation among the three methodologies.
- the maximum relative deviation between the detection results of the 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and those obtained by using the GP73 enzyme-linked immunoassay detection reagent was 30.89%, which was less than 4 times of the average relative deviation between the detection results of the 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and those obtained by using the GP73 enzyme-linked immunoassay detection reagent, namely, 32.36%.
- FIG. 4 a diagram showing the relative bias between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent.
- the maximum relative deviation between the detection results of the 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and those obtained by using the GP73 immunochromatography detection reagent was 33.91%, which was less than 4 times of the average relative deviation between the detection results of the 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and those obtained by using the GP73 immunochromatography detection reagent, namely, 33.98%.
- FIG. 5 a diagram showing the relative bias between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- the maximum relative deviation between the detection results of the 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent and those obtained by using the GP73 immunochromatography detection reagent was 30.48%, which was less than 4 times of the average relative deviation between the detection results of the 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent and those obtained by using the GP73 immunochromatography detection reagent, namely, 30.55%.
- FIG. 6 a diagram showing the relative bias between the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- the relative deviations among the detection results of the three reagents including the GP73 magnetic particle-based chemiluminescence detection reagent, the GP73 enzyme-linked immunoassay detection reagent, and the GP73 immunochromatography detection reagent all fell within the range specified by NCCLS EP9-A, so there was no significant deviation among the three methodologies.
- FIG. 7 shows a linear correlation between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent.
- FIG. 8 shows a linear correlation between the detection results of 200 cases obtained by using the GP73 magnetic particle-based chemiluminescence detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- FIG. 9 shows a linear correlation between the detection results of 200 cases obtained by using the GP73 enzyme-linked immunoassay detection reagent and the detection results of 200 cases obtained by using the GP73 immunochromatography detection reagent.
- the detection reagents adopting three methodologies, namely, the GP73 magnetic particle-based chemiluminescence detection reagent, the GP73 enzyme-linked immunoassay detection reagent, and the GP73 immunochromatography detection reagent had good consistency in regard to the detection results of fatty liver samples.
- the cut-off value of GP73 for use in identification and diagnosis of a simple fatty liver and steatohepatitis in populations with fatty liver was 82.25 ng/ml according to the ROC curve analysis.
- FIG. 10 a ROC curve analysis chart
- FIG. 11 a diagram showing the data distribution of GP73 detection results.
- the serological target marker GP73 and application thereof can be used as an excellent serological target marker for identification and diagnosis of a simple fatty liver and steatohepatitis in populations with fatty liver; when a GP73 content of 82.25 ng/ml was used as a cut-off value for identifying and diagnosing a simple fatty liver and steatohepatitis in populations with fatty liver, the specificity was 86.86%, the sensitivity was 85.71%, and the test results among different detection methods have good consistency.
- the serological target marker GP73 and application thereof are of high clinical application value for clinical identification and diagnosis of a simple fatty liver and steatohepatitis in populations with fatty liver, and for assisting in treatment of a simple fatty liver and steatohepatitis.
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PCT/CN2017/088945 WO2018227643A1 (en) | 2017-06-13 | 2017-06-19 | Target marker gp73 for detecting steatohepatitis and detection application method |
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CN111537719B (en) * | 2020-06-04 | 2022-10-04 | 复旦大学附属中山医院 | Application of serum Sparcl1 protein in non-alcoholic steatohepatitis diagnosis |
CN114858890B (en) * | 2022-04-21 | 2023-08-18 | 桂林电子科技大学 | Method for detecting GP73 based on RGO-CMCS-Hemin/Pt@Pd NPs colorimetric sensor |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040241744A1 (en) * | 2001-07-12 | 2004-12-02 | Hiroaki Kohno | Method of quantifying denatured lipoprotein, reagents for quantifying denatured lipoprotein, method of detecting circulatory disease and reagents for detecting circulatory disease |
US20100184049A1 (en) * | 2007-04-27 | 2010-07-22 | Steve Goodison | Glycoprotein Profiling of Bladder Cancer |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7776550B2 (en) * | 2005-05-05 | 2010-08-17 | Philadelphia Health & Education Corporation | Diagnosis of liver pathology through assessment of protein glycosylation |
WO2009090882A1 (en) * | 2008-01-18 | 2009-07-23 | The University Of Tokyo | Diagnosis method for non-alcoholic fatty liver disease |
JP5322556B2 (en) * | 2008-09-19 | 2013-10-23 | 株式会社Mcbi | Novel nonalcoholic fatty liver disease biomarker and method for detecting nonalcoholic fatty liver disease using the biomarker |
CN101407544B (en) | 2008-11-05 | 2011-12-07 | 曹伯良 | Anti-Golgi apparatus protein monoclonal antibody and use |
CN101735319B (en) * | 2008-11-20 | 2011-10-26 | 北京热景生物技术有限公司 | Monoclonal antibody against GP73 protein, preparation method and application thereof |
WO2012105590A1 (en) * | 2011-02-01 | 2012-08-09 | アステラス製薬株式会社 | Determination marker for non-alcoholic steatohepatitis, and method for diagnosing non-alcoholic steatohepatitis using the marker as measure |
IN2015DN03796A (en) * | 2012-10-17 | 2015-10-02 | Enterome | |
US9469686B2 (en) * | 2013-03-15 | 2016-10-18 | Abbott Laboratories | Anti-GP73 monoclonal antibodies and methods of obtaining the same |
CN103499692B (en) * | 2013-09-26 | 2015-10-14 | 江苏福隆生物技术有限公司 | Golgiosome protein GP 73 quantitative determination reagent kit and detection method thereof |
CN104215761B (en) | 2014-08-27 | 2016-04-20 | 广西医科大学 | Detect the kit of anti-GP73 antibody in serum |
CN106153934B (en) * | 2015-03-26 | 2018-06-19 | 广州瑞博奥生物科技有限公司 | A kind of kit of efficient quantitative detection golgiosome 73 |
CN106290872A (en) * | 2015-05-21 | 2017-01-04 | 天津金虹生物科技开发有限公司 | A kind of golgiosome protein GP 73 quantitative determination reagent kit and detection method thereof |
-
2017
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040241744A1 (en) * | 2001-07-12 | 2004-12-02 | Hiroaki Kohno | Method of quantifying denatured lipoprotein, reagents for quantifying denatured lipoprotein, method of detecting circulatory disease and reagents for detecting circulatory disease |
US20100184049A1 (en) * | 2007-04-27 | 2010-07-22 | Steve Goodison | Glycoprotein Profiling of Bladder Cancer |
Non-Patent Citations (4)
Title |
---|
Torzewski et al., Animal Models of C-Reactive Protein, Hindawl Publishing Corporation, Mediators of Inflammation, vol. 2014, 2014, pp. 1-7. (Year: 2014). * |
Van Der Vekiens et al., Human and equine cardiovascular endocrinology: beware to compare, Cardiovascular Endocrinology 2013, vol. 2, No. 4, pp. 67-76. (Year: 2013). * |
Wei et al., GP73 is a potential marker for evaluating AIDS progression and antiretroviral therapy efficacy, Mol Biol Rep, 2013, 40: pp. 6397-6405. (Year: 2013). * |
Wei et al., Serum GP73 were increased in patients with fatty liver disease, Chinese J Exp Clin Virol, Aug. 2013, vol. 27, No. 4, pp. 244-246. (Year: 2013). * |
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